Fig 1: Loss of MLL and SETD1A affects recruitment of CENPA at centromeres.(A) The representative IF images show the loading of HJURP and CENP-A at the centromere in early G1 phase cells. Parent U-2OS cells (—) or U-2OS cells expressing recombinant full-length MLL (MLL-FL), TAD deleted MLL (MLLΔTAD), and SET domain deleted MLL (MLLΔSET), were treated with either Control or MLL siRNA. Cells, synchronized in the early G1 phase, were stained with α-CENP-A (red) and α-HJURP (green) antibody as shown. (B) Parent U-2OS cells (—) or U-2OS cells stably expressing recombinant full-length SETD1A (SET-FL), and SET domain deleted SETD1A (SETΔSET) were treated with either Control or SETD1A siRNA. (A, B) DNA was stained with DAPI (blue). The area in the white square is magnified and shown on the side of each image. Scale bar, 2 μm. (C–F) Quantification of HJURP and CENP-A fluorescence intensity following depletion of MLL (C, E) and SETD1A (D, F) respectively. ****P ≤ 0.0001, **P ≤ 0.005, ns: not significant, p > 0.05 (Mann–Whitney two-tailed unpaired test). (G) Parent U-2OS cells were treated with either Control or MLL+ SETD1A combined siRNA. Cells, synchronized in the early G1 phase, were stained with α-CENP-A (red) and α-HJURP (green) antibody as shown. Scale bar, 5 μm. (H) Quantification of CENP-A fluorescence intensity following depletion of Control, MLL, SETD1A, or MLL+ SETD1A. ****P ≤ 0.0001, ***P ≤ 0.001, ns: not significant, p > 0.05 (Ordinary one-way ANOVA with Tukey’s multiple comparisons test). Quantification of HJURP intensity for G is shown in S7A Fig. (C–F, H) Each data point represents a single centromere. The error bar represents SEM ≥250 centromeres quantified from 10 early G1 cell pairs, (n = 2 experiments). The raw data underlying parts (C, D) and (E, F, H) can be found in S1 Data. A.U., arbitrary units; MLL, mixed lineage leukemia; SEM, standard error of the mean; SET1, SETD1A; TAD, transcription activation domain.